Nature - USA (2020-08-20)

416 | Nature | Vol 584 | 20 August 2020

Article

activate ISCs after stress^11 ,^13. Six hours of 20HE feeding induced the
Egfr ligands spi and krn and their activating protease rho, but not the
upd2 or upd3 cytokines or Stat signalling (Extended Data Fig. 3a, b).
Exposure to 20HE for 16 h, however, moderately induced upd2, upd3
and Stat activity (Extended Data Fig. 3c–h). The induction of upd2,
upd3 and rho required EcR in ISCs and EBs (that is, ‘progenitors’),
although not in ECs (Extended Data Fig. 3c–e). The Egfr effector
ERK was also mildly activated by 16 h of 20HE exposure, mostly in
progenitors but occasionally in ECs (Extended Data Fig. 3i). ERK
activation required upd2 (Extended Data Fig. 3i), which suggests
a signalling relay^13 ,^14. Notably, the induction of all of these targets
(upd2, upd3, Socs36E, rho, spi and krn) by 20HE was suppressed by
blocking ISC mitoses with RNAi molecules that target string (also
known as stg or cdc25) or Egfr (Extended Data Fig. 3f ). This suggests
that the observed increases in Jak–Stat and Egfr–ERK signalling are
responses to epithelial stress from the early ISC divisions^14. In further
tests, we found that Upd2 from EBs and ECs contributed strongly to
ISC divisions 16 h after 20HE feeding, but only weakly to the early divi-
sions at 6 h (Fig. 1g, Extended Data Fig. 3j–l). Egfr and Rho, however,
were always required (Fig. 1f, Extended Data Fig. 3j–m). We conclude
that ISC divisions are initially activated ISC-autonomously via EcR,
and require Egfr and Rho, whereas later divisions depend in part on
cytokines produced by EBs and ECs, perhaps in response to stress
from the first mitoses. The relationship of EcR to Egfr signalling war-
rants further investigation.
Because mated females produce more ecdysone than virgins or
males^3 ,^5 ,^7 , we tested whether 20HE might account for sex-specific
differences in the gut. Consistent with this, long-term exposure of males

to 20HE phenocopied the female condition, increasing ISC mitoses,

stress responsiveness, epithelial turnover and midgut size (Fig. 1i–k,

Extended Data Fig. 4a–c). Genetically feminizing the male ISCs did not

give these effects (Fig. 1j), which suggests that 20HE acts independently

of genetic sex determination. Forced expression of the ISC mitogen^13

sSpi also failed to enlarge male midguts (Fig. 1j), which indicates that

20HE affects more than just the ISC mitotic rate. Long-term 20HE

feeding also endowed ISCs in virgin females with proliferative char-

acteristics similar to those seen after mating (Extended Data Fig. 4d).

By contrast, RNAi lines that antagonized 20HE signalling in ISCs and

EBs decreased gut size in mated females and suppressed mitoses in

response to detergent stress (Fig. 2c, d, Extended Data Fig. 4e–g). Thus,

sexually dimorphic proliferative traits of ISCs are determined in part

by 20HE signalling.

Similar to human oestrogen and progesterone, ecdysone promotes

behavioural and metabolic changes that enhance female reproduc-

tion^3 ,^7 ,^8. Dose–response assays showed that 1 mM 20HE fed to virgin

females activated EcR targets and ISC mitoses to similar levels to mat-

ing (Fig. 2l, Extended Data Fig. 5a). Hence, we tested whether endog-

enous, mating-induced 20HE activates ISCs. Indeed, mating induced a

large, transient increase in ISC division and enduring gut enlargement^15

(Fig. 2a–d, Extended Data Fig. 5b–h, k). This was independent of genetic

sexual identity (Fig. 2e, Extended Data Fig. 5i). As with exogenously

fed 20HE, these effects initially required EcR only in ISCs, although

EcR in EBs contributed later on (Fig. 2f, g, Extended Data Fig. 5e–j).

Similar to exogenous 20HE, mating also induced expression of upd2

and rho (Extended Data Fig. 5l), which suggests that these are normal

physiological responses.

Vehicle

4 h6 h9 h12 h16 h

0

5

10

15

50

100

150

ISC mitoses per midgut

****NS

****

NS

a esg-Gal4ts (8 days), 16 h feeding

Vehicle1%

SDS

P.e.5 mM

20HE

bcesg-FOts(5 days), 16 h feeding d

Vehicle 5 mM 20HE

Vehicle Vehicle

Vehicle 6 h5 mM 20HE16 h

e

g

20HE 20HE

esg-Gal4ts(8 days),16 h feeding

h esg-Gal4ts(8 days), 16 h feeding

Vehicle P.e.

16 hP.e.

Vehicle6 h20HE16 h20HEVehicle6 h20HE16 h20HE

i esg-FOts (14 days) long-term feeding j esg-Gal4ts(14 days), k

long-term feeding

esg-Gal4ts

(3 days) Su(H)-Gal4

ts

(3 days)

5 mM 20HE, 4-16 h f

Vehicle 5 mM

20HE

esg-Gal4ts, 6 h feeding

Long-term feeding (14 days)

Vehicle 1 mM 20HE

Area = 0.28 mm^2

Length = 3.01 mm

Area = 0.58 mm^2

Length = 4.2 mm

Vehicle

1 mM 20HE

Controltra

RNAi

Controltra

RNAi

Controltra

RNAi

Controltra

RNAi

0

50

100

150

ISC mitoses per midgut

****

NS

**

**

NS

ControlEcR

RNAi

Usp

RNAi

ControlEcR

RNAi

Usp

RNAi

ControlEcR

RNAi

Usp

RNAi

ControlEcR

RNAi

Usp

RNAi

(^200)
4060
80
100
200
300
ISC mitoses per midgut
**** NS ****
ControlRNAi#2EcRControlRNAi#2EcRControlRNAi#2EcRControlRNAi#2EcRControlRNAi#2EcRControlRNAi#2EcR
0
25
50
100
200
300
ISC mitoses per midgut
**** NS****
Controlrho
RNAi
Egfr
RNAi
Controlrho
RNAi
Egfr
RNAi
0
10
20
30
4050
100150
200
ISC mitoses per midgut

---

Controlupd2upd3upd2,3ControlControl Control
0
20
40
100
200
ISC mitoses per midgut

---

---

---

ControlRNAi#1EcRRNAi#2EcRRNAi#1UspRNAi#2UspControlRNAi#1EcRRNAi#2EcRRNAi#1UspRNAi#2Usp
0
25
50
75
100
125
150
ISC mitoses per midgut
NS

---

---

---

NS

---

Vehicle1 mM20HEVehicle1 mM20HE
0
20
40
60
80
100
120
ISC mitoses per midgut

---

NS
Control,vehicleControl,
1 mM 20HE
TraFsSpi
0
0.2
0.4
0.6
0.8
1.0
To tal midgut area (mm
2 )

---

NS NS
DAPIGFP DAPIGFP
DAPIGFP DAPIGFP
DAPIGFP
DAPIGFP
ΔΔΔ
upd2
Δ
upd2,3
Δ
upd2upd3upd2,3
ΔΔΔ
upd2
Δ
upd2,3
Δ
Fig. 1 | Ecdysone induces ISC proliferation and gut growth. a, Midgut mitotic
counts of esg-Gal4ts>UAS-traRNAi virgins after overnight infection with
Pseudomonas entomophila (P.e.), and feeding with 5 mM 20HE or 1% SDS for
16 h. The esg-Gal4ts driver (esg-Gal4 tub-Gal80ts) activates UAS target gene
expression specifically in ISC and EBs (‘progenitor’ cells). b, ISC lineage-tracing
using esgFOts, which drives UAS target gene expression in progenitor cells and
their newborn progeny (ECs or EBs) after a temperature shift. c, Midgut mitotic
counts of esg-Gal4ts>EcRRNAi and esg-Gal4ts>UspRNAi f lies fed 5 mM 20HE for 16 h.
d, Midgut mitotic counts from w^1118 controls fed 5 mM 20HE for different
durations. e, ISC mitoses in midguts expressing EcRRNAi in ISCs and EBs (left) or
in EBs (right) after 6 or 16 h of 5 mM 20HE feeding. f, Mitotic counts in midguts
expressing rhoRNAi or Eg frRNAi in ISCs and EBs 6 h after 20HE feeding. g, Midgut
mitotic counts of upd2Δ, upd3Δ and upd2,3Δ 5–7-day-old mutant and control
f lies after 6 and 16 h of 5 mM 20HE feeding or P.e. infection. h, Mitoses of
EcRRNAi- or UspRNAi-expressing ISCs and EBs after P.e. infection. i, Lineage-tracing
experiment using esgFOts and mitotic counts in 20HE-fed f lies. j, Midgut areas
from male midguts expressing feminizing TraF or sSpi, or fed 20HE for 14 days.
k, Male midgut images. Data are mean ± s.d. NS (not significant), P > 0.05,
P ≤ 0.05, P ≤ 0.01, P < 0.001, ****P < 0.0001, Mann–Whitney (a, c–i) or
ordinary analysis of variance (ANOVA) (j) tests, followed by Bonferroni’s
multiple comparisons test. Exact n values and P values are in the Source Data.
n ≥ 3 independent experiments. Scale bars, 100 μm (b, i) or 1 mm (k). DNA
counterstained blue with DAPI. In all figures, ♂ denotes males, ☿ denotes virgin
females, and ♀ denotes mated females.